BIOREMEDIATION OF PETROLEUM CONTAMINATED BEACH

SEDIMENTS: USE OF CRUDE PALM OIL AND FATTY ACIDS

TO ENHANCE INDIGENOUS BIODEGRADATION
J. P. OBBARD1, , K. L. NG 2 and R. XU1
1

Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4,

Singapore 117576; 2 Tropical Marine Science Institute, 4 Engineering Drive 4, Singapore 117576
( author for correspondence, e-mail: chejpo@nus.edu.sg, Tel: 65-68742884, Fax: 65-67791936)

(Received 13 May 2003; accepted 26 February 2004)

Abstract. Amendment of simple organic carbon, in the presence of inorganic nutrients, to oil contaminated beach sediments can potentially stimulate the biodegradation of hydrocarbons by the indigenous
microbial biomass. The ability of crude palm oil (CPO) and fatty acids, in the presence of soluble
inorganic nutrients (C:N:P = 100:10:1), to stimulate biodegradation in sediments amended with Arabian light crude oil was investigated in laboratory microcosms over a 30-day period. The presence of
nutrients alone enhanced the most probable number (MPN) of hydrocarbon degrading bacteria by almost 12 times after 30 days, and by 26 times in sediment also amended with CPO, thereby confirming
its ability to stimulate the biomass. Addition of individual fatty acids to nutrients-amended sediments
resulted in MPN enhancements of up to 170 times, where straight alkanes (i.e., C17 and C18 ) were
completely degraded. Amendment with CPO, myristic, oleic, linoleic and palmitic acids (0.5% dry
weight equivalent) also enhanced the metabolic activity of the entire microbial biomass, as measured
by intracellular dehydrogenase activity, reaching a maximum of 30 g INTF g dry sed1 h1 . Loss
of branched alkanes was significantly enhanced, where loss of pristane was complete and phytane
loss reached 98% in sediment treated with myristic acid. This study highlights the considerable benefit of adding readily oxidizable fatty acids for enhancing in situ bioremediation of hydrocarbons in
nutrient-amended beach sediments.
Keywords: biodegradation, crude oil, dehydrogenase activity, petroleum hydrocarbons, simple organic carbon source

1. Introduction
Bioremediation can be an effective technology to combat oil pollution on marine foreshore environments (Lee and Merlin, 1999). The technique utilizes microorganisms to reduce the concentration and/or toxicity of a range of chemicals
found in mineral oils (Korda et al., 1997). Biodegradation is a naturally occurring
process where oil-degrading microorganisms are capable, under favorable conditions, of utilizing petroleum hydrocarbons as a metabolic carbon source (Mearns,
1997; Atlas, 1991). Rapid growth in the indigenous microbial biomass can occur in
beach sediments immediately after contamination with oil, but subsequent depletion of nutrients and readily degradable carbon can rapidly curtail biodegradation
rates (Alexander, 1991; Atlas, 1991). Although the application of water-soluble
Water, Air, and Soil Pollution 157: 149161, 2004.
C 2004 Kluwer Academic Publishers. Printed in the Netherlands.


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fertilisers to supply inorganic nutrients can stimulate the microbial biomass, this is
usually short term due to rapid nutrient washout in the open, inter-tidal environment
(Wrenn et al., 1997).
The addition of simple organic carbon sources, in addition to inorganic nutrients, to oil contaminated beach sediments has been recently identified as a useful
technique for further stimulating the biodegradation of petroleum hydrocarbons
by an indigenous microbial biomass. For example, fishmeal and related products
have been found to be superior in stimulating biodegradation compared to the
commercial oleophilic nutrient formulations, such as Inipol EAP-22 (Santas et al.,
1999). Methyl derivatives of vegetable oils (or biodiesel) are also known to stimulate biodegradation and enhance the bioavailability of hydrocarbons in sediments
(Miller and Mudge, 1997; Nelson et al., 1996). Fatty acids are readily degraded
by in situ microorganisms (Ratledge, 1994), and their presence in bioremediation
additives may act as simple carbon sources for microorganisms which then either
co-metabolise petroleum hydrocarbons or switch to hydrocarbon metabolism after depletion (Mudge and Pereira, 1999; Sveum et al., 1994). Enhanced nutrient
immobilisation into the biomass as a result of readily available carbon mineralisation may also reduce nutrient wash-out in the open inter-tidal zone and sustain the
biodegradation process as a result of subsequent biomass attrition and sustained nutrient release (Sveum et al., 1994). Although the entire biomass may not participate
directly in the biodegradation of hydrocarbons, it may serve as a labile reservoir of
nutrients that is slowly released to hydrocarbon degrading bacteria during biomass
attrition and turnover.
This investigation is a follow-up of an earlier field study that successfully demonstrated the inorganic nutrient-enhanced biodegradation of petroleum hydrocarbons
by the indigenous microbial biomass in oil-contaminated, inter-tidal beach sediments in Singapore (Mathew et al., 1999). The prospect of using simple carbon
substrates to further stimulate hydrocarbon degradation was proposed. Vegetable
crude palm oil (CPO) was selected for use on the basis of its rich fatty acid content and its local availability in South East Asia (Palm Oil Research Institute of
Malaysia, 1985; Tan and Oh, 1981). This paper reports on the ability of CPO
and several component fatty acids to stimulate the indigenous microbial biomass
and degrade selected petroleum hydrocarbons in oil-spiked beach sediments under
laboratory conditions as a prelude to further field trials in Singapore.

2. Materials and Methods

2.1. BEACH

SEDIMENT , PREPARATION

The sediment used in this experiment was clean a beach sediment collected from
St. Johns Island, a small island 2 km south of the main island of Singapore.
The sediment was screened to remove particulates greater than 5 mm in size and

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151

adjusted to 15% moisture content using reconstituted seawater (Eaton et al., 1995)
before spiking with an Arabian light crude oil (ALCO) at 5.5 g per 100 g of sand
(dry weight equivalent). The sediment was then weathered for 15 days in darkness
at ambient temperature (i.e., 2334 C) with manual daily mixing and tilling in order to maintain an aerobic condition. After weathering, the oil content of the spiked
sediment decreased to a level of 3.55 g per 100 g of sand (dry weight equivalent) due
to loss of volatile organics. Nutrients were added to sediment in the form of ammonium nitrate, di-sodium hydrogen phosphate, and potassium dihydrogen phosphate
to obtain a molar ratio of C:N:P = 100:10:1. Sediment treatments included
oil-spiked biotic and abiotic controls (i.e., amended with 1% mercuric chloride
solution), and oil-spiked sediments amended with either CPO or single fatty acids
i.e., myristic, oleic, linoleic or palmitic acid, all at 0.5% sediment dry weight
equivalent. All fatty acids, as well as the CPO, no matter if in liquid or solid form,
were physically mixed with sediment and no solvent was added to dissolve or dilute
them. CPO was obtained from a palm-oil processing plant in Johore, Malaysia. All
sediments were prepared in triplicate and placed in aerobic microcosms (500 g of
sediment, dry weight equivalent) at ambient temperature. Nutrients were amended
to all sediments on days 0, 12 and 20, and sediments were tilled every alternate
day to maintain an aerobic condition. Experimental duration was 30 days.
2.2. SEDIMENT

SAMPLING

Twenty grams of sediment (wet weight) was collected from each microcosm on
days 0, 5, 9, 20 and 30 for determination of microbial dehydrogenase activity
(DHA) and GC-MS analysis of selected hydrocarbons. An additional sampling
was undertaken on day 2 for DHA analysis. The most probable number (MPN) of
petroleum hydrocarbon degrading bacteria in sediments was determined on day 0
and day 30, and in the abiotic control 3 h after the addition of 1% mercuric chloride
solution.
2.3. BIOLOGICAL

ANALYSIS

Hydrocarbon degrading microorganisms were enumerated based on a modified

method of Brown and Braddock (1990). Ten mL of 1 M phosphate buffer was added
to 1 g of sediment sample in a sterile conical flask. The mixture was then shaken for
30 min on a reciprocal shaker at 200 rpm. Serial dilutions (i.e., 103 to 106 ) were
then prepared in phosphate buffer and 2 mL aliquots of each dilution were then added
to separate wells of a 24-well tissue culture plate. Each well, containing the aliquot,
was covered with 5 L of filtered-sterilized ALCO. The plates were then incubated
at 30 C and 120 rpm for 21 days. Wells were scored as positive when dispersion
of the oil sheen occurred after incubation. The most probable number (MPN) of
hydrocarbon degrading microorganisms was determined by using a standard MPN
table (Cochran, 1956) and converting counts to the number of cells g dry sed1 .

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The metabolic activity of the entire microbial biomass was determined by measurement of intracellular dehydrogenase activity (DHA) using the method optimized by Mathew and Obbard (2001). DHA is known to be a reliable indicator
of microbial metabolic activity in both soils and sediments (Lee et al., 2000).
2.5 mL deionised water and 1 mL 0.75% freshly prepared 2-(p-iodophenyl)-3-(pnitrophenyl)-5-phenyltetrazoliumchloride (INT) solution (pH 7.9) was added into
5 g (dry weight equivalent) of moist sediment. This sample was incubated in the
dark at 27 C for 22 h, and the metabolic product, INT-formazan (INTF) formed was
extracted by the addition of 25 mL methanol. The extract was then filtered through
a Whatman
R autovial and measured for absorbance at max = 428 nm on a Perkin
Elmer UV-vis Spectrometer Lambda 20. The spectrophotometer was calibrated
with INTF standards prepared in methanol. DHA was expressed as micrograms
INTF g dry sed1 h1 .
2.4. CHEMICAL

ANALYSIS

Loss of oil from sediments was measured by GC-MS analysis of straight (i.e., C17
and C18 indicators) and branched alkanes (i.e., pristane and phytane). The latter
have been used as conservative biomarkers in oil bioremediation studies, but their
recalcitrance has been questioned due to their own susceptibility to biodegradation
(Prince et al., 1994). Therefore, the more stable polycyclic alkane C30 -17(H),
21(H)-hopane was used as the conservative biomarker in this study. This alkane
is insoluble in water, is extremely resistant to biodegradation (Venosa et al., 1997)
and has been used successfully to provide quantitative information on the extent
of hydrocarbon degradation in a range of oil-contaminated environments (Venosa
et al., 1997; Butler et al., 1991).
Sediment samples were dried overnight at 60 C and 5 g of sediment was then extracted with a 165-mL hexane-acetone (1:1, v/v) mixture using soxhlet-extraction.
The extract was then cooled and filtered through grease-free glass microfibre filter
discs (Whatman
R ) into a tared flask (USEPA methods 413.3 and 418.1, 1983). The
filtrate was rotary evaporated (Eyela
R ) for solvent removal at 68.8 C i.e., the boiling point of hexane. The residue was then dissolved in a hexane:acetone mixture
(1:1, v/v) prior to GC-MS analysis. Analysis was undertaken for straight (C12 -C33 )
and branched alkanes (i.e., pristane and phytane), and C30 -17(H), 21(H)-hopane
on a Hewlett-Packard (HP) 6890 GC equipped with a HP 6890 Mass Selective Detector (MSD) and HP6890 auto-sampler. An HP 19091S-433, HP-5MS 5% phenyl
methyl siloxane 30.0 m 250 m i.d. (0.25 m film) capillary column was used for
alkane separation, using helium as the carrier gas at a flow rate of 1.6 mL min1 . The
injector and detector temperatures were set at 290 C and 300 C, respectively. The
temperature program for straight and branched alkanes was set as follows: 2-min
hold at 50 C; ramp to 105 C at 8 C min1 ; ramp to 285 C at 5 C min1 , and 3min hold at 285 C. The temperature program for C30 -17(H), 21(H)-hopane was
set as follows: 2-min hold at 50 C; ramp to 105 C at 8 C min1 ; ramp to 300 C at

BIOREMEDIATION OF PETROLEUM CONTAMINATED BEACH SEDIMENTS

153

5 C min1 , and 5-min hold at 300 C. A 1 L aliquot was injected using a splitless
mode with a 6-min purge-off. The MSD was operated in the scan mode to obtain
spectral data for identification of hydrocarbon components and in the selected ion
monitoring (SIM) mode for quantification of target compounds. Ions monitored
included: alkanes at m/z of 71 and 85; pristane at m/z of 97 and 268; phytane at m/z
of 97 and 282; and hopane at m/z of 191, 177, 412 and 397 (Wang et al., 1994).

3. Results
3.1. MPN

OF PETROLEUM HYDROCARBON DEGRADING MICROORGANISM

MPN data of petroleum hydrocarbon degrading bacteria for the controls and for
various sediment treatments at day 0 and day 30 are given in Table I.
3.2. DEHYDROGENASE

ACTIVITY

DHA levels of the microbial biomass over the 30-day experimental period for the
controls and for various sediment treatments are shown in Figure 1.
3.3. LOSS

OF STRAIGHT AND BRANCHED ALKANES

The ratios of C17 and C18 alkanes to the conservative hopane biomarker for each
of the sediments over 30 days are shown in Figures 2a and 2b, and similarly for
TABLE I
MPN of hydrocarbon degrading bacteria
MPN of bacteria cells (103 )
(cells per gram of dry sediment)
Sediment treatment

Day 0
(a)

Day 30
(b)

Relative change
(b/a)

Control (biotic)
Control (abiotic)
Crude palm oil
Myristic acid
Oleic acid
Linoleic acid
Palmitic acid

75.9
0
75.9
75.9
75.9
75.9
75.9

891.3
0
1 995.3
12 882.5
13 182.6
6 918.3
12 589.3

11.7
0
26.3
169.7
173.7
91.2
165.9

Note. All sediments were amended with inorganic nutrients. Crude palm oil
and fatty acids were added at a concentration of 0.5% (sediment dry weight
equivalent).

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J. P. OBBARD ET AL.

Figure 1. Dehydrogenase activity of the microbial biomass in beach sediments.

phytane and pristane in Figures 3a and 3b. The total percentage losses of C17 and
C18 alkanes, and pristane and phytane are given in Table II.
3.4. S TATISTICAL

ANALYSIS

The results were subjected to analysis of variance (ANOVA) to determine if mean

values of nutrients, n-alkanes, pristane and phytane to hopane ratios, as well as DHA
in the controls and CPO and fatty acids treated sediments differed significantly. Data
were considered to be significantly different between two values if P < 0.01. All
statistical analyses were performed using MINITAB Release 13.2.

4. Discussion
4.1. MPN

OF HYDROCARBON DEGRADING BACTERIA

Approximately 7.6 104 hydrocarbon degrading bacteria per g dry sed1 (Table I)
were present in sediment prior to treatment. Bacteria in the abiotic control were
eliminated after treatment with 1% mercuric chloride and were still absent on day
30. The presence of nutrients alone in the biotic control enhanced the MPN of hydrocarbon degrading bacteria by almost twelve times after 30 days. However, MPN

BIOREMEDIATION OF PETROLEUM CONTAMINATED BEACH SEDIMENTS

155

(a)

(b)
Figure 2. (a) Ratio of straight alkanes (C17 :hopane) for each of the sediments over 30 days. (b) Ratio
of straight alkanes (C18 :hopane) for each of the sediments over 30 days.

was increased by 26 times in sediment that also received CPO (Table I), thereby
confirming the benefits of an additional organic carbon source, in the presence of
inorganic nutrients, on stimulating the population size of indigenous hydrocarbon
degrading bacteria. However, the addition of single fatty acids alone resulted in the

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J. P. OBBARD ET AL.

(a)

(b)
Figure 3. (a) Ratio of branched alkanes (pristine:hopane) for each of the sediments over 30 days. (b)
Ratio of branched alkanes (phytane:hopane) for each of the sediments over 30 days.

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157

TABLE II
Loss of C17 , C18 , pristane and phytane after 30 days. Means and standard deviations of triplicate
samples are showna
Sediment treatment Loss of C17 (%) Loss of C18 (%) Loss of pristane (%) Loss of phytane (%)
Oil-spiked control
(biotic)

77.2 0.9

79.8 1.6

52.0 3.3

36.4 5.4

Oil-spiked control
(abiotic)

2.4 0.1

1.4 1.6

1.0 0.9

0.5 1.5

89.8 1.2

88.9 0.7

87.4 1.0

88.6 1.5

Myristic acid

100.0 0.0

100.0 0.0

100.0 0.0

98.0 0.9

Oleic acid

100.0 0.0

100.0 0.0

100.0 0.0

87.8 1.4

Linoleic acid

100.0 0.0

100.0 0.0

100.0 0.0

83.5 1.0

Palmitic acid

100.0 0.0

100.0 0.0

100.0 0.0

79.6 2.3

Crude palm oil

All sediments were amended with nutrients at a ratio of C:N:P = 100:10:1.

greatest enhancement in the population size of hydrocarbon degrading bacteria. All

fatty acids resulted in an MPN enhancement of approximately 170 times relative
to the biotic control, with the exception of linoleic acid that increased MPN by
approximately 90 times over the duration of the experiment.
4.2. DEHYDROGENASE

ACTIVITY

Microbial DHA in the oil-spiked abiotic control was absent (Figure 1), meaning any
alkane loss in this sediment was solely due to abiotic losses. In contrast, DHA in the
oil-spiked biotic control continued to increase over the duration of the experiment
reaching a peak of approximately 27 g INTF g dry sed1 h1 on day 30. This
increase in metabolic activity was likely a response of the microbial biomass to the
presence of inorganic nutrients in the sediment. Additions of CPO, myristic, oleic,
linoleic and palmitic acids further enhanced DHA relative to the biotic control, at
least up to day 20. Although there was a reduction in DHA in all sediments treated
with fatty acids, apart from palmitic acid, on day 20, levels did not significantly
differ to levels at day 30. For these treatments, DHA increased rapidly and peaked
at approximately 30 g INTF g dry sed1 h1 on day 10, with the exception of sediment treated with palmitic acid where DHA continued to increase to approximately
25 g INTF g dry sed1 h1 on day 30.
4.3. STRAIGHT

ALKANE LOSS

The ratios of the straight alkanes C17 and C18 relative to the stable hopane biomarker
in the abiotic control were almost constant throughout the experiment (Figures 2a
and 2b), meaning that negligible abiotic loss of straight alkanes occurred following

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J. P. OBBARD ET AL.

the initial weathering of oil-spiked sediment (Table II). The slight differences
in C17 and C18 levels between different treatments on Day 0 of the experiment
were probably due to experimental errors and these differences are not significant
(P > 0.05). Losses of straight alkanes, C17 and C18 , in the nutrient-amended biotic
control were significantly greater than the abiotic control (P = 0 and 0.003, respectively), and a total loss of 77.2% of C17 and 79.8% of C18 by day 30 indicates
the beneficial effect of nutrient addition alone. The losses of C17 and C18 in the
sediment treated with CPO after 30 days were not significantly different from the
biotic control (P = 0.011 and 0.019, respectively). The addition of CPO increased
the DHA of the microorganisms, but did not enhance degradation of n-alkanes.
In contrast to CPO amended sediment, C17 and C18 alkanes were completely degraded in sediments treated with individual fatty acids in the presence of inorganic
nutrients (Table II). This enhanced loss is supported by the higher levels of DHA
and the MPN of hydrocarbon degrading bacteria in sediments treated with fatty
acids alone (Table I and Figure 1). The addition of a relatively low amount (i.e.,
0.5%) of fatty acid as a simple carbon source was therefore sufficient to significantly
enhance biodegradation of straight alkanes by hydrocarbon degrading bacteria in
nutrient-amended sediments. The presence of fatty acids provided an easily assimilative alternative growth substrate that stimulated the indigenous microbial
biomass and increased the biodegradation rate of alkanes via co-metabolism. It has
been reported that supplemental carbon additions may allow for mixed substrate
utilization, increases in overall biomass and reduced per-microbe toxin concentrations, hence enhancing biodegradation rates (Baker and Herson, 1994). Sorption
of petroleum hydrocarbons to sediment particles reduces their availability to microorganisms, and hence reduces their biodegradability. The presence of fatty acids
can enhance the biodegradation of n-alkanes as the fatty acids act as nonionic surfactants, which increase the oil-water surface area to improve the bioavailability
of petroleum hydrocarbons for microbial actions (Bruheim et al., 1997; Riis et al.,
2000).
4.4. BRANCHED

ALKANE LOSS

The relative losses of the branched alkanes (i.e., pristane and phytane) in the
nutrient-amended biotic control were significantly lower than in the abiotic control on day 30 (P = 0.001 and 0.003, respectively), with losses of pristane and
phytane at 52% and 36.4% respectively (Table II). In fact, almost no pristane and
phytane was lost throughout the 30-day period in the abiotic control. Biodegradation of pristane and phytane commenced between 5 and 9 days after initiation of
the experiment, and the lower relative loss of phytane compared to pristane is most
likely due to its longer carbon chain length, and hence recalcitrance (Figures 3a
and 3b). Degradation of pristane and phytane in our study confirms their limitation
as biomarkers in hydrocarbon degradation studies due to their own susceptibility
to biodegradation (Prince et al., 1994).

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159

The amendment of CPO as a simple carbon source to sediment, in the presence

of inorganic nutrients, significantly enhanced the degradation of pristane and phytane relative to the biotic control (P = 0.005 and 0.006, respectively), and losses
over 30 days were 87.4% and 88.6% respectively (Table II). Pristane loss was complete in all fatty acid amended sediments. An increase in the degradation rate of
pristane and phytane occurred after day 20 when the rate of C17 , C18 degradation
had slowed and the amount of C17 , C18 left in the sediment was low (Figures 2
and 3). This may represent a metabolic switch in the utilisation of simple to more
complex hydrocarbons by the microbial biomass. The introduction of branching
into the hydrocarbon molecule hinders biodegradation (Baker and Herson, 1994)
and generally, degradation of branched-chain alkanes is repressed by the presence
of straight chain alkanes (DelArco and de Franca, 2001). The effects of different
fatty acids on the biodegradation of phytane were notable, where the loss of phytane
in sediment amended with myristic acid was significantly greater than sediments
treated with linoleic or palmitic acid (Table II; P = 0.004 and 0.009, respectively).
The difference in phytane loss between myristic and oleic acid treatments was not
so significant (P = 0.014). In contrast, the differences between treatments of oleic,
linoleic and palmitic acid were not significant (P > 0.05).
Overall, this study has shown that the amendment of CPO, as a readily available
carbon source, significantly enhanced the population size of hydrocarbon degrading
bacteria and also increased the metabolic activity of the entire indigenous microbial biomass in the oil contaminated sediments under laboratory conditions. The
addition of CPO to further enhance biodegradation under tropical conditions is
therefore beneficial for a bioremediation program. This is consistent with recent
studies that have highlighted the beneficial effects of providing an additional carbon
source to the indigenous microbial biomass (Santas et al., 1999; Miller and Mudge,
1997; Nelson et al., 1996). However, the addition of myristic, oleic, linoleic and
palmitic fatty acids, in the presence of inorganic nutrients, resulted in higher rates
of biodegradation of straight and branched alkanes. Sveum et al. (1994) showed
that the supply of external carbon in additives was important for the assimilation
and retention of essential nutrients in microbial biomass, thereby reducing nutrient
loss in an open beach environment. The presence of fatty acids may also accelerate
the biodegradation rate of crude oil due to their surfactant properties, and their
ability to enhance the bioavailability and dispersion of hydrocarbons (Riis et al.,
2000; Nelson et al., 1996). Indeed, a combination of nutritional and surfactant properties in bioremediation additives is recognised as an advancement in petroleum
hydrocarbon remediation (Santas et al., 1999).
CPO represents a more complex carbon source than individual fatty acids, and
in our study this may have resulted in a delay in hydrocarbon loss compared to
sediments treated with component fatty acids alone, as well as a less beneficial
effect on the MPN of hydrocarbon degrading bacteria and the DHA of the microbial
biomass. Although Sveum et al. (1994) showed that microorganisms metabolised
both fish-meal additive and hydrocarbons simultaneously in the bioremediation of

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oil-spiked sediments, albeit at different rates, a switch to hydrocarbon metabolism

was dependent on the total amount of additive supplied to the sediment.

5. Conclusion
In this study, all the fatty acids (i.e., myristic, oleic, linoleic or palmitic acid) and
CPO, as a supplementary carbon source, significantly enhanced the MPN and DHA
of microbial biomass. All fatty acids also significantly elevated the biodegradation
of n-C17 , n-C18 , pristane, and phytane in oil-contaminated sediments amended with
nutrients relative to a biotic and abiotic control (P < 0.01), where myristic acid
was the most effective among all the fatty acids. CPO significantly enhanced the
biodegradation of pristane and phytane (P < 0.01). The addition of simpler component fatty acids alone is more effective in stimulating oil biodegradation rates
than CPO. Since fatty acids can act as both a co-substrate and a nonionic surfactant (Bruheim et al., 1997; Riis et al., 2000), further investigations are planned to
determine their effects on the biodegradation rates of polycyclic aromatic hydrocarbons (PAHs) which are less bioavailable and more recalcitrant than alkanes in
oil-contaminated sediments.

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